Transcriptional analysis of prebiotic uptake and catabolism by Lactobacillus acidophilus NCFM

Abstract

The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β-linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.

Figure 1. Representative volcano plots of the oligosaccharide-induced differential global transcriptome within L. acidophilus NCFM.

All genes are shown as black dots (·) and all statistically significant upregulated genes involved with oligosaccharide metabolism (Table 1) are depicted as white circles (○).

Figure 2. Phenotypic characterization of single gene deletions within L. acidophilus NCFM.

Growth profiles are shown on galactose (A) and raffinose (B and C) for the mutants within the stachyose-induced gene cluster lacking the GH36 α-galactosidase ΔLBA1438 (Δ) or the solute binding protein component of the ABC transporter ΔLBA1442 (○) compared to upp-wildtype (•).

Figure 3. Organization of gene clusters encoding upregulated genes by potential prebiotic oligosaccharide stimulation.

All genes are listed with locus tag number and gene name (PTS permeases are shown with domain name; regulators, hypothetical proteins and transposons are abbreviated as reg, hyp. and trans respectively). Gene product functions are colored red for glycoside hydrolases, light grey for transcriptional regulators, blue for PTS permease domains, dark grey for proteins unrelated to carbohydrate metabolism, green for ABC transporter domains and yellow for the GPH permease. All upregulated genes (Table 2) are shown with framed boxes, CRE regulatory sites are represented by arrows and predicted rho-independent transcription terminators by stem loops.

Figure 4. Reconstructed uptake and catabolic pathways in L. acidophilus NCFM.

Proteins are listed by locus tag LBA numbers, transporters are colored by class (Figure 3) and glycoside hydrolases are listed with GH family number. The polydextrose fraction transported by the ABC transporter (LBA0502–LBA0505) is uncertain and thus the hydrolytic pathway is marked as unknown. The present data outlines the PTS permease LBA0606 (higher level of induction compared to LBA0502–LBA0505) and associated hydrolytic pathway, as the main route of polydextrose utilization by L. acidophilus NCFM.